3D printed model of Cas9 from CRISPR

We need to talk about CRISPR

By Gesa Junge, PhD

You’ve probably heard of CRISPR, the magic new gene editing technique that will either ruin the world or save it, depending on what you read and whom you talk to? Or the Three Parent Baby, which scientists in the UK have created?

CRISPR is a technology based on a bacterial immune defense system which uses Cas9, a nuclease, to cut up foreign genetic material (e.g., viral RNA). Scientists have developed a method by which they can modify the recognition part of the system, the guide RNA, and make it specific to a site in the genome that Cas9 then cuts. This is often described as “gene editing” which allows disease-causing genes to be swapped out for healthy ones.

CRISPR is now so well known that Google finally stopped suggesting I may be looking for “crisps” instead, but the real-world applications are not so well worked out yet, and there are various issues around CRISPR, including off-target effects, and also the fact that deleting genes is much easier than replacing them with something else. But, after researchers at Oregon Health and Science University managed to change the mutated version of the MYBPC3 gene to the unmutated version in a viable human embryo last month, the predictable bioethical debate was reignited, and terms such as “Designer Babies” got thrown around a lot.

A similar thing happened with the “Three Parent Baby,” an unfortunate term coined to describe mitochondrial replacement therapy (MRT). Mitochondria, the cells’ organelles for providing energy, have their own DNA (making up about 0.2% of the total genome) which is separate from the genomic DNA in the nucleus, which is the body’s blueprint. Mitochondrial DNA can mutate just like genomic DNA, potentially leading to mitochondrial disease, which affects 1 in 5000-10000 children. Mitochondrial disease can manifest in various ways, ranging from growth defects to heart or kidney to disease to neuropsychological symptoms. Symptoms can range from very mild to very severe or fatal, and the disease is incurable.

MRT replaces the mutated mitochondrial DNA in a fertilized egg or in an embryo with the healthy version provided by a third donor, which allows the mitochondria to develop normally. The UK was the first country to allow the “cautious adaption” of this technique.

While headlines need to draw attention and engage the reader for obvious reasons, oversimplifications like “gene editing” and dramatic phrases like “three parent babies” can really get in the way of broadening the understanding of science, which is difficult enough as it is. Research is a slow and inefficient process that easily gets lost in a 24-hour news cycle, and often the context is complex and not easily summed up in 140 characters. And even when the audience can be engaged and interested, the relevant papers are probably hiding behind a paywall, making fact checking difficult.

Aside from difficulties communicating the technicalities and results of studies, there is also often a lack of context in presenting scientific studies - think for example of chocolate and red wine which may or may not protect from heart attacks. What is lost in many headlines is that scientific studies usually express their results as a change in risk of developing a disease, not a direct causation, and very few diseases are caused by one chemical or one food additive. On this topic, WNYC’s “On The Media”-team have an issue of their Breaking News Consumer Handbook that is very useful to evaluate health news.

The causation vs. correlation issue is perhaps a little easier to discuss than big ethical questions that involve changing the germline DNA of human beings because ethical questions do not usually have a scientific answer, let alone a right answer. This is a problem, not just for scientists, but for everyone, because innovation often moves out of the realm of established ethics, forcing us to re-evaluate it.

Both CRISPR and MRT are very powerful techniques that can alter a person’s DNA, and potentially the DNA of their children, which makes them both promising and scary. We are not ready to use CRISPR to cure all cancers yet, and “Three Parent Babies” are not designed by anyone, but unfortunately, it can be hard to look past Designer Babies, Killer Mutations and DNA Scissors, and have a constructive discussion about the real issues, which needs to happen! These technologies exist; they will improve and eventually, and inevitably, play a role in medicine. The question is, would we rather have this development happen in reasonably well-regulated environments where authorities are at least somewhat accountable to the public, or are we happy to let countries with more questionable human rights records and even more opaque power structures take the lead?

Scientists have a responsibility to make sure their work is used for the benefit of humanity, and part of that is taking the time to talk about what we do in terms that anyone can understand, and to clarify all potential implications (both positive and negative), so that there can be an informed public discussion, and hopefully a solution everyone can live with.

 

Further Reading:

CRISPR:

National Geographic

Washington Post

 

Mitochondrial Replacement Therapy:

A paper on clinical and ethical implications

New York Times (Op-Ed)

 


CRISPR gene editing in human embryos

Engineering Babies One Crispr at a Time

 

By Sophie Balmer, PhD

Over the past few weeks, the scientific community has been overwhelmed with major advances in human embryonic research. Whether researchers report for the second time the use of Crispr to edit the human germline or extend the conditions of in vitro culture of human embryos (also here), these issues have been all over the news. However, as all topics can not be raised in only one post, therefore, I will focus on genome editing studies.

 

About a year ago, one research group in China reported the first genome editing of human embryos using Crispr technology. Although these embryos were not viable due to one additional copy of each chromosome, this study quickly became highly controversial and raised strong concerns. The public and scientific communities questioned whether editing the human germline for therapeutic benefits was legitimate, leading to numerous ethical discussions. A few of weeks ago, a second study reported genome editing of embryos reinforcing the debate around this issue. Additionally, several research proposal involving genomic modification of healthy human embryos’ DNA have been validated recently in other countries. In this post, I want to address several questions. What are the possible advances or consequences of such work? What is the current legislation on human genome editing worldwide? Are these studies as alarming as what is written in some newspaper articles?

 

The emergence of the Crispr technology a few years ago has revolutionized the way scientists work since this method greatly improves the efficiency of DNA alteration of model organisms. However, this powerful tool has also raised many concerns, notably on the possibility to easily tweak the human genome and generate modified embryos.

In the eyes of the general public, this kind of experiment resonates with science fiction books or movies. Because of the high potential of this technique, it is crucial to inform everyone correctly to avoid clichés. Recently, one of my favorite comedian and television host John Oliver depicted in a very bright and amusing way how small scientific advances are sometimes presented in the media. Although the examples he uses are dramatic, every scientific breakthrough gets its share of overselling to the public. In the case of gene-editing of human embryos, pretending we are about to use eugenics principles to engineer babies and their descendants with beneficial genes is pure fiction. However, to prevent any potential malpractice from happening, clear ethical discussions and regulations need to be established and then explained to the public to prevent misunderstanding of these issues.

Within the scientific community, last year’s results triggered the need for new discussions and regulations on human cloning. Modifying the genome of human embryos involves modifying the germline as well, leading eventually to the transmission of the genetic alteration to future generations. However, the consequences of such transmission are unknown. Potentially, this could resolve a number of congenital genetic diseases for the individual him/herself and be used for gene therapy but would result in generations of genetically modified humans.

 

Because of cultural and ethical differences between countries, the legislation (if there is any) around working with human embryos or cells derived from human embryos (hESC for human embryonic stem cells) is variable. International ethical committees have only been able to establish guidelines as instituting international laws on human cloning is impossible. Ultimately, each country is responsible for enforcing these rules. Most countries and international ethics committees agree on a ban on reproductive and therapeutic human cloning. Moreover, following last year published experiments, a summit held in December 2015 gathered experts from all around the world. The consortium concluded that gene-editing of embryos used to establish pregnancy should not be performed (for now) and to follow up on all-related issues, new sets of guidelines are coming out imminently.

 

Still, it seems difficult to get an idea of the consensus depending on the countries in which scientists perform experiments. There is range of possibilities when working with human samples: some countries completely prohibit any manipulation of human embryos or hESC while others authorize genetic modification of the embryo for research purposes only under specific conditions. In between several nations authorize research exclusively on already derived lines of hESC and others authorize derivation of hESC but no manipulation of the embryos themselves.

Besides these general rules and as of today, three countries have approved proposals for gene-editing of human embryos: China, the UK and Sweden. Research proposals in both European countries have authorized Crispr targeting of specific genes in healthy human embryos to assess the function of these genes during early human development. However, these embryos can not be used for in vitro fertilization (IVF) and have to be destroyed at the end of the study. The purpose of these studies would be to confirm what has been described in hESC and in mammalian model systems and contribute to our knowledge of human development.

 

On the other hand, both published studies from China focused on Crispr targeting towards clinical therapies of an incurable blood disease or HIV. The overall purpose of such projects is to test the use of the Crispr technology for gene therapy. Although rendering embryos immune to several diseases using Crispr is an attractive possibility, it seems more urgent to probe the validity of the technique in humans and assess whether the mechanisms of human embryonic development are similar to what has been hypothesized. Gene therapies have already been successfully attempted in humans using other techniques to modify the genome. Yet, the modifications were targeted towards specific cells in already-born individuals. Again, modifying the genome of embryos implies that the mutation will be inherited in future generations and is in a large part the reason of this debate. Moreover, Crispr targeting still leads to unspecific modification of the genome, although very promising results show that newly engineered cas9 could lead to very specific targeting. The consequences of such off-target modification are unknown and could be disastrous for the following generations.

 

Overall, no research proposal dares to consider genetically modified embryos to establish pregnancy but as research moves faster, increasing demand for ethical discussion and regulations are brought forward. As more studies come out, it will be interesting to follow the evolution of this debate. Additionally, informing clearly the population of the possibilities and outcomes of ongoing projects should be a priority so that they can give an informed consent towards such research. In any case, a clear boundary needs to be established between selecting the fittest embryo by pre-implantation genetic diagnosis, which is routinely performed for IVF and playing the sorcerer’s apprentice with human embryo’s


Fighting Zika Virus with Mosquito Genetics

 

By  John McLaughlin

 

The Zika virus burst into the news last year when a dramatic increase in microcephaly cases was reported throughout several states in Brazil. This frightening birth defect quickly became associated with the mosquito-borne virus, carried by Aedes mosquitos; Aedes aegypti, which also carries Dengue, is the main vector in the current Zika outbreak. While Zika virus usually affects adults with fairly mild symptoms such as fever, rash, and joint pain, it can have severe or fatal consequences for the fetuses being carried by infected females. In fact, The World Health Organization (WHO) has recently reported a scientific consensus on the theory that Zika is the cause of the large number of Brazilian microcephaly cases.

 

In January of 2016, a Hawaiian baby born with microcephaly became the first case of Zika reported in the United States. And the U.S. National Institute of Allergy and Infectious Diseases has recently stated that a wider outbreak of the virus within the United States will likely occur soon. Naturally, mosquito containment has become a top priority for health officials in both infected areas and those likely to be impacted by the virus. The standard list of mosquito control protocols includes pesticide repellents, mosquito nets, eliminating stagnant open water sources, and long-sleeved clothing to limit skin exposure. In addition to these, health authorities are considering a number of new strategies based on genetic engineering technologies.

 

One such technique employs the concept of gene drive, the fact that some “selfish” gene alleles can segregate into gametes at frequencies higher than the expected Mendelian ratios. In this scenario, gene drive can be exploited to spread a disease resistance gene quickly throughout a population of mosquitoes. Recently, a team at the University of California tested this idea by using CRISPR technology to engineer the mosquito Anopheles stephensi with a malarial resistance gene drive. After integration of the resistance gene cassette and DNA targeting with CRISPR, this gene was successfully copied onto the homologous chromosome with high efficiency, thus ensuring that close to 100% of its offspring will bear resistance. Possibly, similar techniques could be exploited to engineer Zika resistance in Aedes mosquitoes.

 

In contrast to engineering disease resistance, an alternative defense strategy is to simply reduce the population of a specific mosquito species, in the case of a Zika outbreak, Aedes aegypti. The WHO has recently approved a GM mosquito which, after breeding, produces offspring that die before reaching adulthood. This technique can dramatically reduce an insect population when applied in strategic locations. The British biotech firm Oxitech has also developed its own strain of sterile Aedes aegypti males. In laboratory testing, these GM mosquitoes compete effectively with wild males for female breeding partners. The short-term goal is receiving approval to test these sterile males in the wild; ultimately, a targeted release of these mosquitoes will reduce the Aedes aegypti population in Zika hot spots without affecting other species.

 

In parallel to mosquito engineering, other work has focused on studying the mechanisms underlying Zika’s dramatic affects on the brain. To study the process of Zika infection in vitro, scientists at Johns Hopkins cultured 3-D printed brain organoids and demonstrated that the virus preferentially infects neural stem cells, resulting in reduced cortical thickness owing to the loss of differentiated neurons. This neural cell death may explain the frequent microcephaly observed in fetuses carried by infected mothers.

 

Much like the recent outbreak of Ebola in several African countries, this event helps underscores the importance of basic research. A recent New York Times article drew attention to this fact by highlighting the need for more complete genome sequences of the mosquito species that carry Zika. With a complete genome sequence at hand, researchers might be able to piece together information in answering questions such as: why are some Aedes mosquitoes vectors for Zika and others aren’t? Species differences in genome sequence may provide some answers. Nevertheless, greater knowledge of the mosquito’s biology will yield more options for human intervention. This is an excellent case study in how ‘basic’ and ‘translational’ research projects can co-evolve in special situations.

 


DNA bases

CRISPR/Cas9: More Than a Genome Editor

By Rebecca Delker, PhD

 

The bacterial defense system, CRISPR/Cas9, made huge waves in the biomedical community when the seemingly simple protein-RNA complex of Type II CRISPR systems was engineered to target DNA in vitro and in complex eukaryotic genomes. The introduction of double-strand breaks using CRISPR/Cas9 in a targeted fashion opened the portal to highly affordable and efficient site-specific genomic editing in cells derived from yeast to man.

 

To get a sense of the impact CRISPR technology has had on biological research, one simply needs to run a search of the number of publications containing CRISPR in the title or abstract over the past handful of years; the results practically scream in your face. From 2012, the year of the proof-of-principle experiment demonstrating the utility of engineered Cas9, to 2015, CRISPR publications rose steadily from a mere 138 (in 2012) to >1000 (at the time of this post). Publications more than doubled between the years of 2012 and 2013, as well as between 2013 and 2014. Prior to the use of CRISPR as a technology, when researchers studied the system for the (very cool) role it plays in bacterial defense, publications-per-year consistently fell below 100. In other words, it’s a big deal.

 

In fact, during my 10 years at the bench I have never witnessed a discovery as transformative as CRISPR/Cas9. Overnight, reverse genetics on organisms whose genomes were not amenable to classical editing techniques became possible. And with the increasing affordability of high-throughput sequencing, manipulation of the genomes of non-model organisms is now feasible. Of course there are imperfections with the technology that require greater understanding to circumvent (specificity, e.g.), but the development of CRISPR as a tool for genomic engineering jolted biological research, fostering advances more accurately measured in leaps rather than steps. These leaps – and those expected to occur in the future – landed the discoverers of CRISPR/Cas9 at the top of the list of predicted recipients of the Nobel Prize in Chemistry; though they didn’t win this year (the award went to researchers of the not-totally-unrelated field of DNA repair), I anticipate that a win lies ahead. The rapid success of CRISPR genome editing has also sparked patent battles and incited public debate over the ethics of applying the technology to human genomes. With all of the media attention, it’s hard not to know about CRISPR.

 

The transformative nature of CRISPR/Cas9 does not, however, end with genome editing; in fact, an even larger realm of innovation appears when you kill the enzymatic activity of Cas9. No longer able to cut DNA, dead Cas9 (dCas9) becomes an incredibly good DNA-binding protein guided to its target by a programmable RNA molecule (guide RNA, gRNA). If we think of active Cas9 as a way to better understand genes (through deletions and mutations), then dCas9 is the route to get to know the genome a bit better – a particularly enticing mission for those, including myself, invested in the field of Genomics. From high-throughput targeted gene activation and repression screens to epigenome editing, dCas9 is helping scientists probe the genome in ways that weren’t possible before. Here, I put forth some of the best (in my humble opinion) applications, actual and potential, of CRISPR technology that go beyond genome editing.

 

Cas9 and Functional (Epi)Genomics

 

For many years the genome was considered as the totality of all genes in a cell; the additional junk DNA found was merely filler between the necessary gene units, stitching together chromosomes. We’ve come a long way since this naiveté, especially in recent years. We understand that the so-called junk DNA contains necessary regulatory information to get the timing and position of gene expression correct; and now, more than ever, we have a greater appreciation for the genome as a complex macromolecule in its own right, participating in gene regulation rather than acting as a passive reservoir of genetic material. The genome, it has been shown, is much more than just its sequence.

 

The epigenome, consisting of a slew of modifications to the DNA and the histones around which the DNA is wrapped, as well as the 3D organization of the genome in the nucleus, collaborates with DNA binding proteins to accurately interpret sequence information to form a healthy, functional cell. While mutations and/or deletions can be made – more easily, now, with Cas9 – to genomic sequences to test functionality, it is much harder to conduct comparable experiments on the epigenome, especially in a targeted manner. Because of the inability to easily perturb features of the epigenome and observe the consequences, our understanding of it is limited to correlative associations. Distinct histone modifications are associated with active versus inactive genes, for example; but, how these modifications affect or are affected by gene expression changes remains unknown.

 

Taking advantage of the tight binding properties of dCas9, researchers have begun to use the CRISPR protein as a platform to recruit a variety of functionalities to a genomic region of interest. Thus far, this logic has most commonly been employed to activate and/or repress gene expression through recruitment of dCas9 fused to known transcriptional activator or repressor proteins. Using this technique, scientists have conducted high-throughput screens to study the role of individual – or groups of – genes in specific cellular phenotypes by manipulating the endogenous gene locus. And, through a clever extension of the gRNA to include a hairpin bound by known RNA-binding proteins, the targeted functionality has been successfully transferred from dCas9 to the gRNA, allowing for simultaneous activation and repression of independent genes in the same cell with a single dCas9 master regulator – the beginnings of a simple, yet powerful, synthetic gene circuit.

 

Though powerful in its ability to decipher gene networks, dCas9-based activation and repression screens are still gene-centric; can this recruitment technique help us better understand the epigenome? The first attempts at addressing this question used dCas9 to target histone acetyltransferase, p300, to catalyze the acetylation of lysine 27 on histone 3 (H3K27) at specific loci. The presence of H3K27 at gene regulatory regions has been known to be strongly associated with active gene expression at the corresponding gene(s), but the direction of the histone modification-gene expression relationship remained in question. Here, Hilton et al. demonstrate that acetylation of regulatory regions distal to gene promoters strongly activates gene expression, demonstrating causality of the modification.

 

More recently, recruitment of a dCas9-KRAB repressor fusion to known regulatory regions catalyzed trimethylation of lysine 9 on histone 3 (H3K9) at the enhancer and associated promoters, effectively silencing enhancer activity. Though there have only been a few examples published, it will likely not be long until researchers employ this technique for the targeted analysis of additional epigenome modifiers. Already, targeted methylation, demethylation and genomic looping have been accomplished using the DNA-binders, Zinc Finger Nucleases and TALEs. With the increased simplicity in design of gRNAs, dCas9 is predicted to surpass these other proteins in its utility to link epigenome modifications with gene expression data.

 

Visualization of Genomic Loci

 

When you treat dCas9 as a bridge between DNA and an accessory protein, just as in the recruitment of activators, repressors and epigenome modifiers, there are few limits to what can be targeted to the genome. Drawing inspiration from the art of observation that serves as the foundation of scientific pursuit, researchers have begun to test whether dCas9 can be used to visualize genomic loci and observe their position, movements, and interactions simply by recruiting a fluorescent molecule to the locus of interest.

 

This idea, of course, is not entirely new. In situ hybridization techniques (ISH, and its fluorescent counterpart, FISH) have been successfully used to label locus position in fixed cells but cannot offer any information about the movement of chromosomes in living cells. Initial studies to conquer this much harder feat made use of long tracts of repetitive DNA sequence bound by its protein binding partner fused to fluorescing GFP; though surely an advance, this technique is limited because of the requirement to engineer the repetitive DNA motifs prior to imaging.

 

To circumvent this need, researchers have recently made use of TALEs and dCas9 (and here) carrying fluorescent tags to image unperturbed genomic loci in a variety of live cell cultures. The catch is that both TALEs and dCas9 perform much better when targeting repetitive regions, such that multiple copies of the fluorescent molecule are recruited, enhancing the intensity of the signal. Tiling of fluorescent dCas9 across a non-repetitive region using 30-70 neighboring gRNAs (a task made much more feasible with CRISPR versus TALEs) can similarly pinpoint targeted loci, albeit with much higher background. As is, the technique lacks the resolution desired for live imaging, but current advances in super-resolution microscopy and single-molecule tracking, as well as improvements in the brightness of fluorescent molecules available, will likely spur improvements in dCas9 imaging in the coming years.

 

Finally, dCas9 is not only useful in live cells. CASFISH, an updated Cas9-mediated FISH protocol, has been successfully used to label genomic loci in fixed cells and tissue. This updated version holds many benefits over traditional FISH including a streamlined protocol; but, most notably, CASFISH does not require the denaturation of genomic DNA, a necessary step for the hybridization of FISH probes, eliminating positional artifacts due to harsh treatment of the cells. Unfortunately, as of now, CASFISH also suffers from a need for repetitive sequences or tiling of gRNAs to increase signal intensity at the locus of interest.

 

Targeting RNA with Cas9

 

From cutting to tagging to modifying, it is clear that Cas9 has superstar potential when teamed up with double-stranded DNA (dsDNA); however, recent data suggests that this potential may not be limited to DNA. Mitchell O’Connell and colleagues at Berkeley found that Cas9 could bind and cleave single-stranded RNA (ssRNA) when annealed to a short DNA oligonucleotide containing the necessary NGG sequence. In addition, the authors made use of dCas9 and biotin-tagged gRNA to capture and immobilize targeted messenger RNA from cell extract. Though it remains to be shown, this proof-of-principle binding of dCas9 suggests that it is plausible to recruit a variety of functionalities to RNA as has been done for dsDNA. Recruitment of RNA processing factors through Cas9 could potentially enhance translation, generate known RNA editing events (deamination, e.g.), regulate alternative splicing events, or even allow visualization of RNA localization with conjugated fluorescent molecules. Again, each of these processes requires no modification to the RNA sequence or fixation, both of which can disrupt normal cell physiology.

 

Improving CRISPR Technology

 

The development of CRISPR technology, particularly the applications discussed here, is still in its infancy. It will likely take years of research for Cas9 and dCas9 to reach their full potential, but advances are underway. These developments pertain not only to the applications discussed here, but also genome engineering.

 

Specificity of Cas9

 

Cas9’s biggest flaw is its inability to stay focused. Off-target (OT) binding (and here) of Cas9 and DNA cutting have been reported and both present problems. With particular relevance to dCas9-based applications, promiscuous binding of Cas9 to regions of the genome that contain substantial mismatches to the gRNA sequence raises concerns of non-specific activity of the targeted functionality. Efforts to reduce OT binding are needed to alleviate these concerns, but progress has been made with the finding that truncated gRNA sequences are less tolerant of mismatches, reducing off-target Cas9 activity, if not also binding.

 

Temporal Precision of Cas9

 

One of the most exciting developments in dCas9 genome targeting is the potential to manipulate the genome and epigenome in select cell populations within a whole animal to gain spatial resolution in our understanding of genome regulation; however, as we have learned over the years, gene expression patterns don’t only change with space, but also time. A single cell, for example, will alter its transcriptome at different points during development or in response to external stimulus. The development of split versions of Cas9 (and dCas9), which require two-halves of the protein to be expressed simultaneously for function, will not only improve spatial specificity of Cas9 activity but holds the potential to restrict its activity temporally. Drug-inducible and photoactivatable (!) versions of split Cas9 restrict function to time windows of drug treatment or light activation, respectively. In addition, a ligand-sensitive intein has been shown to temporally control Cas9 activity by releasing functional Cas9 through protein splicing only in the presence of ligand.

 

Expanding the CRISPR Protein Repertoire

 

Finally, CRISPR technology will likely benefit from taking all of the weight off of the shoulders of Cas9. Progress toward designing Cas9 molecules with altered PAM specificity, as well as the isolation of Cas9 from different species of bacteria, has helped expand the collection of genomic sites that can be targeted. It has also enabled multiplexing of orthogonal CRISPR proteins in a single cell to effect multiple functions simultaneously. More recently, the Zhang lab isolated an alternative type II CRISPR protein, Cpf1, purified from Francisella novicida. Cas9’s new BFF is also able to cut genomic DNA (as shown in human cells), but in a slightly different fashion than Cas9, generating sticky overhangs rather than blunt ends. Cpf1 also naturally harbors an alternate PAM specificity; rather than targeting sequences upstream of NGG, it prefers T-rich signatures (TTN), further expanding the genomes and genomic sites that can be targeted.

 

CRISPR/Cas9 has already proven to be one of the most versatile tools in the biologist’s toolbox to manipulate the genomes of a variety of species, but its utility continues to grow beyond these applications. Targeting Cas9 to the mitochondria rather than the nucleus can specifically edit the mitochondrial genome, with implications for disease treatment. Cas9 has been used for in vitro cloning experiments when traditional restriction enzymes just won’t do. And, by directly borrowing the concept of Cas9 immunity from bacteria, researchers have enabled enhanced resistance to viruses in plants engineered with Cas9 and gRNAs. While we ponder what innovative technique will come next, it’s important to think about how this cutting-edge technology that promises to bolster both basic and clinical research came to be: this particular avenue of research was paved entirely by machinery provided by the not-so-lowly bacteria. That’s pretty amazing, if you ask me.


DNA gel

Biotech Breakthrough: The CRISPR/Cas System

 

By John McLaughlin

In the last few years, a huge amount of excitement has grown over the CRISPR/Cas system and its use in targeted genome editing; this acronym derives from Clustered Regularly Interspaced Short Palindromic Repeats and their CRISPR-associated genes (Cas). CRISPR loci, which are found in many species of bacteria and most archae, have been collectively described as an RNA-based “immune system,” because of their ability to recognize and destroy foreign phage and plasmid DNA.

 

Although the acronym was first coined in a 2002 paper, CRISPR has only recently been exploited as a research tool. How does the system work and what is its use in the lab? There are at least three distinct types of CRISPR system. A typical “type II” CRISPR locus consists of several protein-coding Cas genes adjacent to an array of direct repeat and spacer sequences. The direct repeats are usually palindromic and conserved, in contrast to the much more variable spacers; these repeat-spacer sequences are transcribed as one unit and then processed into short CRISPR-RNAs (crRNAs).  A 2007 Science article demonstrated that a bacterial population could acquire resistance to phage infection by incorporating DNA fragments from the invading phage genome into a CRISPR locus, in the form of new spacer sequences. The newly acquired spacers are then transcribed and processed into crRNAs, associate with a trans-activating RNA (tracRNA) and Cas protein, and are eventually guided to a homologous DNA sequence to catalyze a double-stranded break.

 

The CRISPR system can be flexibly “reprogrammed” by designing custom chimeric RNAs (chiRNA), which serve the function of both crRNA and tracRNA in one molecule. By co-expressing a “designer” chiRNA with a Cas protein, a targeted and specific DNA break can be created in the genome; after providing an exogenous DNA template to help repair the break, customized knock-ins or knock-outs can be generated. Judging from the rapid technical advances made in the last few years, the system promises to be an efficient and high-throughput format for genome editing. To date, knock-outs have been created in a variety of organisms including rats, flies, and human cells.

 

CRISPR/Cas technology has attracted scientific attention as well as commercial interests. In November 2014, biologists Jennifer Doudna and Emmanuelle Charpentier were honored as co-recipients of the 2015 Breakthrough Prize in the Life Sciences, for their work in dissecting the mechanism of CRISPR’s sequence-specific DNA cleavage. According to its proponents, the possible applications of the CRISPR system seem almost limitless. CRISPR Therapeutics, a recently formed company dedicated to translating the technology into genetic disease therapies, has raised 25 million dollars from new investors. And just last month, the pharmaceutical company Novartis began collaborations with Intellia Therapeutics and Caribou Biosciences in order to pursue new therapeutics using CRISPR/Cas.

 

A technology as potentially lucrative as this one does not develop without controversy. MIT Technology Review recently reported on the competing startup companies aiming to exploit CRISPR technology, and the ensuing battles over intellectual property rights in different organisms. In fact, last year the Broad Institute and MIT were awarded a patent which covers the use of CRISPR genome-editing technology in eukaryotes. Feng Zhang, who is listed as Inventor on the patent, and his lab at MIT were the first to publish on CRISPR’s functionality in human cells.

 

In a few years, this exciting technology may be a commonplace fixture of the biology lab. Only time will tell if the CRISPR craze produces the amazing breakthroughs that scientists, and the general public, are eagerly awaiting.